Prolonged use by women of oral contraceptives containing the synthetic ethinyl estradiol (EE) is associated with a modest increased risk of developing liver tumors. Our original hypothesis was that the non- directly mutagenic EE was a promoter of hepatocarcinogenesis. Since then, our laboratory and others demonstrated that EE is a strong promoter and weak complete carcinogen of hepatocarcinogenesis in female rats, confirming this hypothesis. During the previous project period (years 6-9) we observed that at non-hepatotoxic doses, following the initial transient stimulation of growth, continued EE exposure was associated with the onset of a mitosuppressed state, characterized by reduced basal growth and decreased responsiveness to growth stimulation. Our overall hypothesis was/is that mitosuppression represents a growth- negative, selective environment and that the altered hepatic foci that develop represent clonal outgrowths of hepatocytes that became resistant to mitosuppression through spontaneous or carcinogen-induced mutagenesis ("initiation"). In other words, initiated hepatocytes would be differentially resistant to EE-induced mitosuppression. Our hypothesis during the current project period (years 10-14) was that mitosuppression was caused by EE-induced altered gene expression. At the time the project began (January 1995), using differential display, we had just detected several cDNAs representing mRNA transcripts increased in amount during EE-induced mitosuppression. These transcripts, and others, originate from nuclear and mitochondrial genome-encode genes encoding proteins of the respiratory chain. The induction by EE of these transcripts in human hepatoma HepG2 cells, cultured rat hepatocytes and cultured precision-cut liver slices requires estrogen metabolism, most likely to catechols, and the estrogen receptor. In vivo and in culture, the increase in mitochondrial transcripts precedes an increase in mitochondrial superoxide production, reflecting increased respiratory chain activity. We do not known whether this response is mechanistically related to mitosuppression, although EE inhibited basal and transforming growth factor beta (TGF-induced apoptosis in cultured hepatocytes and liver slices. We hypothesize that mitosuppression reflects reduced hepatocyte growth/turnover due to the inhibition of apoptosis by EE. We further hypothesize that the inhibition of apoptosis results from EE catechol metabolites signaling through the estrogen receptor to stimulate mitochondrial respiration. Since mitochondria are integral to some signaling processes leading to induction of apoptosis, we propose that increased mitochondrial respiration renders hepatocytes less sensitive to apoptosis inducing signals. During the next project period (years 15-20) we would investigate these hypotheses through 3 Specific Aims. 1) To determine the mechanism of inhibition of apoptosis by EE; 2) to determine whether altered hepatic foci are resistant to inhibition of apoptosis by EE, and 3) To define in detail the signal transduction pathway mediating the induction by EE of mitochondrial respiratory chain activity.